截短型Fibrobacter succinogenes 1,3-1,4-β-D- 葡聚醣水解酶突變種E85I的晶體結構

Crystal Structure of Truncated Fibrobacter succinogenes 1,3-1,4-β-D-glucanase Mutant E85I

江元能(Yuan-Neng Chiang)

指導教授 ： 蔡麗珠

國立臺北科技大學/工程學院/有機高分子研究所/碩士(2008年)

https://doi.org/10.6841/NTUT.2008.00578

摘要

Fibrobacter succinogenes1,3-1,4-β-D-葡聚醣水解酶（Fsβ-glucanase、lichenase、EC 3.2.1.73）屬於醣苷水解酶第16號家族，它是一種存在於牛胃中的寄生細菌所分泌的酵素，能有效與特異性的水解穀物中的β-D-葡聚糖（β-D-glucan）或地衣聚醣（lichenan）中與β-1,3鍵連接之β-1,4的鍵結位置，而形成三至五個寡醣組成的最終產物。我們以懸滴蒸氣擴散法培養得到截短型1,3-1,4-β-D-葡聚醣水解酶突變種E85I的晶體，再利用X-ray繞射儀收集分析的數據得知空間族群（space group）為P3121的三方晶系（trigonal），三邊長為a = b = 69.6 Å和c = 97.4 Å ; 三夾角為α=β= 90˚和γ= 120˚，解析度為2.2 Å。此突變種E85I的蛋白質摺疊方式與野生種非常相似，具有β-三明治結構（jellyroll β-sandwich）。除了發現在胺基酸Asn164、Asn189、Gly222的位置有鈣離子和二個水分子形成八面體的幾何形狀外，在E85I突變種中還有另一個銫離子與胺基酸Ser155、Leu157和三個水分子形成三角型雙金字塔的鍵結。且發現位在凹槽面的四個 loops 有往外邊移動的現象，位於凹槽面活性區域內的胺基酸殘基Glu60也有移轉的現象。從動力學之數據結果得知，將帶有負電荷的麸胺酸換成非極性的異白胺酸，水解催化能力下降了約3倍，故經由此結構和動力學之數據得知Glu85扮演影響醣類催化功能的重要角色。

關鍵字

1,3-1,4-β-D-葡聚醣水解酶突變種E85I ； 分子取代法 ； 蒸氣擴散法

並列摘要

Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase, EC 3.2.1.73) belongs to glycosyl hydrolases family 16. Fsβ-glucanase specifically hydrolyzes the β-1,4-glycosidic bonds when β-1,3-glycosidic linkages are located prior to β-1,4-glycosidic bond in lichenan or β-D-glucans. It has been suggested that the truncated Fsβ-glucanase could accommodate five glucose rings into its active site upon enzyme-substrate interaction and many residues involved with substrate binding. Glu85 was proposed to make stacked interaction with subunit +1 of carbohydrate substrate in our previous study of enzyme complex structure. Here we describe the mutant E85I crystal structure to a 2.2 Å resolution. The overall structure of E85I mutant was similar to the jellyroll β-sandwich structure of the wild type enzyme. One Ca2+ ion and one Cs+ ion were found on the surface of the E85I structure with six- and five- ligands, respectively. Further analysis of the E85I mutant structure revealed that the catalytic residue Glu60 shifted away because the loop located at the concave site moved approximately 2 Å from its position in the native enzyme complex. The E85I structure and kinetic data suggest that Glu85 plays an important role in catalytic function.

並列關鍵字

1,3-1,4-β-D-glucanase ； mutante E85I ； molecular replacement

參考文獻

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